REVIEW
published: 16 October 2020
Edited by:
Giovanna Batoni,
University of Pisa, Italy
Reviewed by:
Cesar de la Fuente-Nunez,
University of Pennsylvania,
United States
Paula Gomes,
University of Porto, Portugal
*Correspondence:
Qing Kong
kongqing@ouc.edu.cn;
kongqingg@163.com
Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Received: 13 July 2020
Accepted: 23 September 2020
Published: 16 October 2020
Citation:
Huan Y, Kong Q, Mou H and Yi H
(2020) Antimicrobial Peptides:
Classification, Design, Application
and Research Progress in Multiple
Fields. Front. Microbiol. 11:582779.
doi: 10.3389/fmicb.2020.582779
Frontiers in Microbiology | www.frontiersin.org
doi: 10.3389/fmicb.2020.582779
Antimicrobial Peptides:
Classification, Design, Application
and Research Progress in Multiple
Fields
Yuchen Huan, Qing Kong* , Haijin Mou and Huaxi Yi
College of Food Science and Engineering, Ocean University of China, Qingdao, China
Antimicrobial peptides (AMPs) are a class of small peptides that widely exist in nature
and they are an important part of the innate immune system of different organisms.
AMPs have a wide range of inhibitory effects against bacteria, fungi, parasites and
viruses. The emergence of antibiotic-resistant microorganisms and the increasing of
concerns about the use of antibiotics resulted in the development of AMPs, which
have a good application prospect in medicine, food, animal husbandry, agriculture and
aquaculture. This review introduces the progress of research on AMPs comprehensively
and systematically, including their classification, mechanism of action, design methods,
environmental factors affecting their activity, application status, prospects in various
fields and problems to be solved. The research progress on antivirus peptides, especially
anti-coronavirus (COVID-19) peptides, has been introduced given the COVID-19
pandemic worldwide in 2020.
Keywords: antimicrobial peptides, classification, coronavirus, mode of action, design, motifs, application
INTRODUCTION
Alexander Fleming discovered lysozyme in 1922, and this discovery marked the birth of modern
innate immunity. Since then, antibiotics and antimicrobial peptides (AMPs) have been discovered.
A total of 3,240 AMPs have been reported in the antimicrobial peptide database (APD31 ) updated
on August 24, 2020.
Different types of AMPs have the following commonalities: their number of amino acid residues
is between 10 and 60 (average: 33.26), and almost all AMPS are cationic (average net charge: 3.32).
However, several anionic AMPs also exist, and they have several acidic amino acids like aspartic
acid and glutamic acid (Malkoski et al., 2001; Schittek et al., 2001; Lai et al., 2007).
The anti-microbial resistance of microorganisms is becoming increasingly serious with the
abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing
countries. Research from Kenya has detected substantial amounts of antibiotic residues in edible
meat (Ayukekbong et al., 2017). The prevalence of vancomycin-resistant Enterococcus (VRE)
and methicillin-resistant Staphylococcus aureus (MRSA) is increasing in clinical medicine, so the
countermeasures are urgently needed to address these bacterial infections. However, from the
1
http://aps.unmc.edu/AP/
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Huan et al.
perspective of pharmaceutical companies, the development of
new antibiotic drugs results in low profit. Thus, replacing
antibiotics has become a consideration in the pharmaceutical,
agricultural, animal husbandry, and food industries.
Research on AMPs is continuously developing and
considerable amounts of data on AMPs have been stored in
AMP databases. However, the mechanism of AMPs remains
incompletely understood, and further work needs to be
performed to determine the relationship between different
physicochemical properties to obtain low-cost and highly safe
AMPs with remarkable antimicrobial effects and the specificity
and a high capacity for synergies of AMPs should also be further
developed (Lazzaro et al., 2020).
CLASSIFICATION OF AMPs
The diversity of natural AMPs causes difficulty in their
classification. AMPs are classified based on (1) source,
(2) activity, (3) structural characteristics, and (4) amino
acid-rich species (Figure 1).
Classification of AMPs Based on
Sources
The sources of AMPs can be divided into mammals (human host
defense peptides account for a large proportion), amphibians,
microorganisms, and insects according to statistical data in
APD3. The AMPs found in oceans have also attracted
widespread attention.
Mammalian Antimicrobial Peptides
Mammalian antimicrobial peptides are found in human, sheep,
cattle, and other vertebrates. Cathelicidins and defensins are the
main families of AMPs. Defensins can be divided into α-, β-, and
θ-defensins depending on the position of disulfide bonds (Reddy
et al., 2004). Human host defense peptides (HDPs) can protect
human from microbial infections but show different expressions
in every stage of human growth. For example, cathelicidin
LL-37, a famous AMP derived from the human body, is usually
detected in the skin of newborn infants, whereas human beta-
defensin 2 (hBD-2) is often expressed in the elderly instead of
the young (Gschwandtner et al., 2014). HDPs can be identified
in many parts of the body such as skin, eyes, ears, mouth,
respiratory tract, lung, intestine, and urethra. Besides, AMPs in
human breast milk also play an important role in breastfeeding
because it can decrease the morbidity and mortality of breast-
feeding infants (Field, 2005). What’s interesting is that Casein201
(peptide derived from β-Casein 201–220 aa), identified in
colostrum, shows different levels in preterm human colostrum
and term human colostrums (Zhang et al., 2017). Dairy is an
important source of AMPs, which are generated through milk
enzymatic hydrolysis. Several AMPs have been identified from
α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions,
and the most famous peptide obtained is lactoferricin B (LfcinB)
(Sibel Akalın, 2014). Furthermore, whether the AMPs derived
from dairy products can be used for dairy preservation is also an
interesting subject to develop.
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Antimicrobial Peptides: Classification, Design, Application
In addition to antimicrobial activity, HDPs, such as
cathelicidins and defensins, also affect immune regulation,
apoptosis, and wound healing (Wang, 2014).
Amphibian-Derived Antimicrobial Peptides
Antimicrobial peptides from amphibians play an important role
in the protection of amphibians from the pathogens that have
induced the global amphibian population decline (Rollins-Smith,
2009). Frogs are the main source of amphibian AMPs and the
most famous AMP from frogs is magainin; the skin secretions
of frogs from genera Xenopus, Silurana, Hymenochirus, and
Pseudhymenochirus under the Pipidae family are rich in AMPs
(Conlon and Mechkarska, 2014). Furthermore, cancrin, which
has an amino acid sequence of GSAQPYKQLHKVVNWDPYG,
has been reported as the first AMP from the sea amphibian
Rana cancrivora (Lu et al., 2008). This marks a broader source
of AMPs of amphibians.
Insect-Derived Antimicrobial Peptides
Antimicrobial peptides are mainly synthesized in fat bodies and
blood cells of insects, which is one of the main reasons for insects’
strong adaptability to survival (Vilcinskas, 2013). Cecropin is the
most famous family of AMPs from insects, and it can be found
in guppy silkworm, bees, Drosophila. Cecropin A shows activity
against different inflammatory diseases and cancers (Dutta et al.,
2019). What should be known is that the number of AMPs varies
greatly between species, for example, invasive harlequin ladybird
(Harmonia axyridis) and black soldier fly (Hermetia illucens)
have up to 50 AMPs, while pea aphid (Acyrthosiphon pisum)
lacks AMPs (Shelomi et al., 2020). Jellein, a peptide derived from
bee royal jelly, shows promising effects on several bacteria and
fungi, and its lauric acid-conjugated form can inhibit the parasite
Leishmania major (Zahedifard et al., 2020).
Microorganisms-Derived Antimicrobial Peptides
Antimicrobial peptides can be obtained from microorganisms
like bacteria and fungi, and some famous peptides are nisin,
gramicidin from Lactococcus lactis, Bacillus subtilis, and Bacillus
brevis (Cao et al., 2018). Due to the high price of chemical
synthesis of AMPs, the biological expression has attracted the
increase of attention. Specific yeast species like Pichia pastoris,
Saccharomyces cerevisiae, and bacteria like Escherichia coli,
B. subtilis, and plants have been used for expression systems
(Parachin et al., 2012), but it should be noticed that because of
the toxicity, proteolytic degradation, and purification, AMPs are
difficult to be produced in E. coli, which is necessary to take
advantage of fusion tags (Yu et al., 2015).
Besides, numerous AMPs have also been extracted and
isolated from the stems, seeds, and leaves of plants, and they
are classified into several groups, including thionins, defensins
and snakins (Tang et al., 2018). More marine-derived AMPs
have been reported to have given the increasing value allotted
by people to marine resources. Although most of the reported
marine AMPs have been tested in vitro, several of these AMPs
have shown promising results in vivo, for example, As-CATH4
shows an immunity-stimulating effect in vivo and can enhance
the anti-infective capability of drugs used in combination with it
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Huan et al. Antimicrobial Peptides: Classification, Design, Application

FIGURE 1 | Classification of antimicrobial peptides.

(Semreen et al., 2018). Myticusin-beta is an immune-related AMP Antifungal Peptides (AFPs)

of Mytilus coruscus and a promising alternative to antibiotics
(Oh et al., 2020). Moreover, GE33, known as pardaxin, is a
marine AMP and the GE33-based vaccine has shown the ability
to enhance antitumor immunity in mice (Huang et al., 2013).
Antifungal peptides are a subclass of AMPs that address
fungal infections with enhanced drug resistance. Many AFPs
have shown excellent anti-fungal activities against common
pathogenic fungi, such as Aspergillus and Candida albicans
in clinical medicine, yeast, filamentous fungi (e.g., Aspergillus

Classification Based on Activity

The activity of AMPs can be divided into 18 categories according
to the statistics of the ADP3 database. These categories can be
summarized as antibacterial, antiviral, antifungal, antiparasitic,
anti-human immunodeficiency virus (HIV), and anti-tumor
peptides (Figure 2).

Antibacterial Peptides
Antibacterial peptides account for a large part of AMPs
and have a broad inhibitory effect on common pathogenic
bacteria, such as VRE, Acinetobacter baumannii, and MRSA
in clinical medicine and S. aureus, Listeria monocytogenes,
E. coli in food and Salmonella, Vibrio parahaemolyticus in
aquatic products. Many natural and synthetic AMPs like nisin,
cecropins and defensins have shown good inhibition activity to
Gram-positive bacteria and Gram-negative bacteria. In recent FIGURE 2 | Statistics of the main functions of antimicrobial peptides.
research, AMPs P5 (YIRKIRRFFKKLKKILKK-NH2 ) and P9 Antibacterial peptides account for the largest proportion, approximately 60%,
followed by antifungal peptides, which account for 26%, and antiviral,
(SYERKINRHFKTLKKNLKKK-NH2 ), which are designed based
antiparasitic, anticancer, anti-HIV peptides account for almost the same about
on Aristicluthys nobilia interferon-I, inhibit MRSA and show a 2–5% (the figure is drawn based on data in APD3).
low cytotoxicity (Li C. et al., 2019).

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Huan et al.
flavus), mold in food and agriculture. Except for brevinin,
ranatuerin, cecropins, many synthetic peptides also show good
antifungal activity. For example, AurH1, derived from aurein
1.2, can effectively treat C. albicans infection, which has a lethal
rate up to 40% (Madanchi et al., 2020). Aflatoxin, which is a
carcinogen produced by A. flavus, is harmful to the human
body. Many AFPs can inhibit the growth of A. flavus. For
example, an AFP with a sequence of FPSHTGMSVPPP can
inhibit the growth of A. flavus MD3. A total of 37 antifungal
peptides isolated from Lactobacillus plantarum TE10 and their
mixture can reduce A. flavus spore formation in fresh maize seeds
(Muhialdin et al., 2020). Moreover, two chemically synthesized
radish AMPs show a good inhibitory effect against different yeast
species, such as Zygosaccharomyces bailii and Zygosaccharomyces
rouxii (Shwaiki et al., 2020).
Antiviral Peptides (AVPs)
Viruses cause serious harm to human life and huge economic
losses to the animal husbandry. The COVID-19, which is the
recent outbreak, has caused great loss of lives and properties.
Furthermore, foot-and-mouth disease virus, avian influenza virus
(AIV), and HIV are long-term threats to human life. So, it is
extremely urgent to solve these problems, and antiviral peptides
provide new ways. Antiviral peptides show a strong killing effect
on viruses mainly by (1) inhibiting virus attachment and virus
cell membrane fusion, (2) destroying the virus envelope, or
(3) inhibiting virus replication (Jung et al., 2019) (shown in
Figure 3). A recent report has shown that AMP Epi-1 mediates
the inactivation of virus particles and has good inhibitory activity
against foot-and-mouth disease virus (Huang et al., 2018).
Moreover, infectious bronchitis virus (IBV) is the pathogen of
infectious bronchitis and the inoculation of swine intestinal AMP
FIGURE 3 | Examples of specific targets for Antiviral peptides.
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Antimicrobial Peptides: Classification, Design, Application
(SIAMP)–IBV mixed solution remarkably reduced the mortality
of chicken embryos compared with the IBV infection group,
showing the good inhibitory activity of SIAMP on IBV (Sun et al.,
2010). Anti-HIV peptides are a subclass of anti-viral peptides.
The most important examples of these peptides include defensins
(including α- and β-defensins, which have different mechanisms),
LL-37, gramicidin D, caerin 1, maximin 3, magainin 2,
dermaseptin-S1, dermaseptin-S4, siamycin-I, siamycin-II, and
RP 71955 (Madanchi et al., 2020) and antiviral peptide FuzeonTM
(enfuvirtide) has been commercialized as an anti-HIV drug
(Ashkenazi et al., 2011).
Due to the global spread of the COVID-19 (Figure 4A),
the antiviral peptides against the coronavirus will be discussed
in more detail. Coronaviruses (CoVs) belong to the family
Coronaviridae; they are enveloped viruses with a positive-sense
single-stranded RNA genome and have a helical symmetry
(Franks and Galvin, 2014). CoVs, including severe acute
respiratory syndrome CoV (SARS-CoV) and Middle East
respiratory syndrome coronavirus (MERS-CoV) (Mustafa et al.,
2018), and the recent outbreak of COVID-19 have caused
serious threats to human life and property. CoVs can cause life-
threatening respiratory diseases and the viral particle is formed
by spike glycoprotein (S), the envelope (E), the membrane
(M), and the nucleocapsid (N) (Vilas Boas et al., 2019). It
should be noted that their infectivity requires viral spike
(S) protein. Fusion inhibitor peptides combine with the S
protein to interfere with its folding and prevent infection.
Besides, the S2 domain of the SARS-CoV S protein contains
heptad repeat HR1 and HR2 sequences. Peptide HR2 (HR2:
SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL) and its
lipid-binding peptide is highly similar or even identical to the
near-membrane portion of S protein ferredoxin, which interferes
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Huan et al.
FIGURE 4 | Information of COVID-19. (A) C-F13-nCoV Wuhan strain 02,
Strain Number: CHPC 2020.00002; NPRC 2020.00002, Source: National
Pathogen Resource Collection Center (National Institute for Viral Disease
Control and Prevention under Chinese Center for Disease Control and
Prevention). (B) Structure of novel coronavirus spike receptor-binding domain
complexed with its receptor ACE2. (DOI: 10.2210/pdb6LZG/pdb).
with refolding into post-fusion fusion-catalyzing domains (FDs)
(Du et al., 2009; Park and Gallagher, 2017). According
to recent research, the lipopeptide EK1C4, derived from
EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL), is
the most effective fusion inhibitor against COVID-19 S protein-
mediated membrane fusion (Xia et al., 2020). Homology
modeling and protein-peptide docking showed that temporin
has potential therapeutic applications against MERS-CoV
(Marimuthu et al., 2019). Two AMPs from the non-structural
protein nsp10 of SARS-CoV, K12, and K29, can inhibit SARS-
CoV replication (Ke et al., 2012). Furthermore, rhesus theta-
defensin 1 (RTD-1) treated animals have a marked reduction in
mortality in the presence of SARS-CoV while the peptide alone
shows airway inflammation and the one possible mechanism
of action for RTD-1 is immunomodulatory (Wohlford-Lenane
et al., 2009). In general, AMPs against coronavirus can be
roughly classified as i) peptides derived from HR1, HR2 and
RBD subunits of the spike protein, ii) peptides derived from
other AMPs, iii) Peptides derived from non-structural protein
(Mustafa et al., 2018). Furthermore, molecular docking analysis
indicated that peptides were employed to disrupt the interaction
between COVID-19 and ACE2 (angiotensin-converting enzyme
2) to inhibit COVID-19 entrance in cells (Figure 4B) (Souza
et al., 2020). Finally, it should be noted that this therapy lacks
clinical trials and the main method of animal experiments
is an intranasal administration. This reminds us that nasal
drug delivery (NDD) is a potential therapy for AMPs as
anti-coronavirus drugs. Besides, the antiviral database AVPdb2
includes numerous antiviral peptides.
Antiparasitic Peptides
Parasitic protozoa can cause diseases in human and animals
through a variety of routes, including animal-to-person or
person-to-person contact, water, soil, and food (Chalmers et al.,
2020). And with the increase in parasite drug resistance, the
need for new treatments has increased. Antiparasitic peptides
show their killing effect on parasites which cause diseases such
2
http://crdd.osdd.net/servers/avpdb/
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Antimicrobial Peptides: Classification, Design, Application
as malaria and leishmaniasis (Mangoni et al., 2005; Rhaiem
and Houimel, 2016) and AMPs like cathelicidin, temporins-
SHd show high inhibition activity against parasites (Abbassi
et al., 2013). In recent research, Epi-1, a marine synthetic AMP,
can remarkably inhibit Trichomonas vaginalis by destroying its
membrane (Neshani et al., 2019). The peptide Jellein derived
from bee royal jelly which has introduced above and 4-amino
acid AMP KDEL (lysine, aspartic acid, glutamic acid, and leucine)
has shown a significant effect on the Leishmania parasite (Cao
et al., 2019; Zahedifard et al., 2020). However, it should be noted
that their mechanisms are not the same. Cyanobacterial peptides
differ from higher-eukaryote AMPs because their antiparasitic
action depends on specific protein targets. Thus, these target
parasites can be distinguished accurately even though they belong
to the same family or genus (Rivas and Rojas, 2019).
Anticancer Peptides (ACPs)
The ACPs show anticancer mechanisms by (1) recruiting
immune cells (such as dendritic cells) to kill tumor cells, (2)
inducing the necrosis or apoptosis of cancer cells, (3) inhibiting
angiogenesis to eliminate tumor nutrition and prevent
metastasis, and (4) activating certain regulatory functional
proteins to interfere with the gene transcription and translation
of tumor cells (Wu D. et al., 2014; Ma et al., 2020). Tritrpticin
and its analogs induce considerable toxicity toward Jurkat cells
in vitro, whereas indolicidin and puroindoline A can also act as
ACPs (Arias et al., 2020). It should be noted that both net charge
and hydrophobicity play important roles in optimizing the
anticancer activity of ACPs and they can constrain and influence
each other. Thus, achieving a balance between net charge and
hydrophobicity is important for better anticancer activity.
Besides the peptide mentioned above, anti-inflammatory,
anti-diabetic peptides, spermicidal peptides etc. have been
noticed, but they are not the same as antimicrobial peptides.
Simply put, anti-inflammatory peptides decrease the release
of inflammatory mediators and inflammatory cytokines (nitric
oxide, interleukin-6, and interleukin-1β) and some of them also
inhibit inflammatory signals like NF-κB, MAPK, and JAK-STAT
pathways (Meram and Wu, 2017; Gao et al., 2020). Anti-diabetic
peptides play their function by modulating the G protein-
coupled receptor kinase (GRK 2/3) or activating glucagon-
like peptide-1 (GLP-1), glucagon receptors (Marya et al., 2018;
Graham et al., 2020). However, it is not accurate to classify
these types of peptides as AMPs and bioactive peptides may be
more convincing.
Classification of AMPs Based on Amino
Acid-Rich Species
Proline-Rich Peptides (PrAMPs)
Proline is a typical non-polar amino acid. PrAMPs behave
differently from other AMPs, that is, they enter bacterial
cytoplasm by the inner membrane transporter SbmA instead
of killing bacteria through membrane destruction (Mattiuzzo
et al., 2007). Once in the cytoplasm, PrAMPs target ribosomes
and block the binding of aminoacyl-tRNA to peptidyltransferase
center or trap decoding release factors on the ribosome during
the termination of translation to interfere with protein synthesis
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Huan et al.
(Seefeldt et al., 2015). For instance, Tur1A, which is an
orthologous AMP of bovine PrAMP Bac7 discovered from
Tursiops truncatus, interferes with the transition from the initial
phase to the extension phase of protein synthesis by binding to
ribosomes. In addition, different PrAMPs lack a high sequence
similarity but have short motifs containing repeating proline
and arginine (Arg) residues (e.g., -PPXR- in Bac5 and -PRPX-
in Bac7) (Mardirossian et al., 2018, 2019). Although PrAMPs
mainly kill Gram-positive bacteria, pPR-AMP1, a proline-rich
AMP identified from crab (Scylla paramamosain), exhibits
antimicrobial activity against Gram-positive and Gram-negative
bacteria (Imjongjirak et al., 2017). Besides, pieces of research
have shown that PrAMPs have immunostimulation activity
(Li W. et al., 2016).
Tryptophan- and Arginine-Rich Antimicrobial
Peptides
Tryptophan (Trp), as a non-polar amino acid, has a remarkable
effect on the interface region of the lipid bilayer, whereas Arg, as
a basic amino acid, confers peptide charge and hydrogen bond
interactions, which are essential properties to combine with the
bacterial membrane’s abundant anionic component. And it seems
that Trp residues play the role of natural aromatic activators of
Arg-rich AMPs by ion-pair-π interactions (Walrant et al., 2020),
thereby promoting enhanced peptide-membrane interactions
(Chan et al., 2006). In addition to indolicidin and Triptrpticin
which both are famous AMPs that rich in Arg and Trp residues.
Octa 2 (RRWWRWWR) is also a typical Trp- and Arg-rich AMP
that inhibits Gram-negative E. coli and Pseudomonas aeruginosa
and Gram-positive S. aureus. And short Trp- and Arg-rich AMPs
designed based on bovine and murine lactoferricin have also
shown strong inhibitory action against bacteria (Strøm et al.,
2002; Bacalum et al., 2017).
Histidine-Rich Peptides
Histidine is a common basic amino acid, and histidine-rich AMPs
show good membrane permeation activity. HV2 is a histidine-
rich AMP designed based on RR(XH)2 XDPGX(YH)2 RR–NH2
(where X represents I, W, V, and F). This peptide increases
the permeability of bacterial cell membranes to cause cell
membrane rupture and death. In addition, HV2 inhibits bacterial
movement in a concentration-dependent manner and shows
a strong anti-inflammatory effect by inhibiting the production
of tumor necrosis factor α (TNF-α) (Dong et al., 2019). An
AMP designed based on Octa 2 has shown good therapeutic
potential by replacing its Arg residues with histidine (Bacalum
et al., 2017). Furthermore, L4H4, which is designed based
on the linear cationic amphiphilic peptide magainin, also
shows good antibacterial activity and cell penetration properties
by inserting four histidine sequences in leucine and alanine
(Lointier et al., 2020).
Glycine-Rich Antimicrobial Peptides
The R group of glycine is generally classified as a non-polar
amino acid in biology. Glycine-rich AMPs, such as attacins and
diptericins, widely exist in nature (Lee et al., 2001; Kwon et al.,
2008). These peptides contain 14% to 22% glycine residues, which
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Antimicrobial Peptides: Classification, Design, Application
have an important effect on the tertiary structure of the peptide
chain. A glycine-rich AMP derived from salmonid cathelicidins
activates phagocyte-mediated microbicidal mechanisms, which
differ from the mechanism of conventional AMPs (D’Este et al.,
2016). Furthermore, the glycine-rich central–symmetrical GG3
is an ideal commercial drug candidate against clinical Gram-
negative bacteria (Wang et al., 2015).
Classification Based on Antimicrobial
Peptide Structures
Antimicrobial peptides can be divided into four categories based
on their structures including linear α-helical peptides, β-sheet
peptides, linear extension structure, and both α-helix and β-sheet
peptides (Figure 5) (Lei et al., 2019). Moreover, progressively
cyclic peptides and AMPs with more complex topologies
(including lasso peptides and thioether bridged structures) are
reported (Koehbach and Craik, 2019).
ANTIMICROBIAL PEPTIDE ACTION
MECHANISM
Membrane Targeting Mechanism
The membrane-targeting mechanisms of AMPs can be described
through models, including the pole and carpet models and the
pole model can be further divided into the toroidal pore and
barrel-stave models (Figure 6).
The Toroidal Pore Model
The toroidal pore model is also known as the wormhole
model. In this model, AMPs vertically embedded in the cell
membrane accumulate and then bend to form a ring hole with
a diameter of 1–2 nm (Matsuzaki et al., 1995, 1996). The typical
examples of this model are magainin 2, lacticin Q, and arenicin.
Furthermore, cationic peptides, including TC19, TC84, and BP2,
compromise the membrane barrier by creating fluid domains
(Omardien et al., 2018).
Barrel-Stave Model
Antimicrobial peptides aggregate with each other, penetrate the
bilayer of the cell membrane in the form of multimers, and
form channels that result in the cytoplasmic outflow. In severe
cases, AMPs can induce cell membrane collapse and lead to cell
death (Lohner and Prossnigg, 2009). For instance, Alamethicin
performs its pore-forming activity by using this model. Besides,
hairpin AMP protegrin-1 can form stable octameric β-barrels and
tetrameric arcs (half barrels) in implicit and explicit membranes
by simulations (Lipkin and Lazaridis, 2015).
Carpet-Like Model
Antimicrobial peptides are arranged parallel to the cell
membrane. Their hydrophilic end faces the solution, and their
hydrophobic end faces the phospholipid bilayer. AMPs will cover
the membrane surface that similar to a carpet and destroy the
cell membrane in a ‘detergent’-like manner (Oren and Shai,
1998). However, this pore-forming mechanism requires a certain
concentration threshold and the required concentration of AMPs
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FIGURE 5 | Different structures of AMPs. (A) LL-37 adopts a typical α-helical conformation (DOI: 10.2210/pdb2K6O/pdb). (B) Gomesin is a β-sheet peptide and
stabilized by disulfide bonds (DOI: 10.2210/pdb1KFP/pdb). (C) Indolicidin is a AMP with linear extension structure instead of well-defined 3D structure (DOI:
10.2210/pdb1G89/pdb). (D) α1-purothionin adopts both alpha-helix and beta-sheet conformation, and arrows indicate extension direction (DOI:
10.2210/pdb2plh/pdb).

FIGURE 6 | Models of action for extracellular AMP activity. (A) Carpet model: accumulation of AMPs on the surface and then destroy the cell membrane in the
manner of “detergent”. (B) Barrel stave model: AMPs aggregate with each other and are inserted into the bilayer of the cell membrane in the form of multimers and
arrange parallel to the phospholipids, then form a channel. (C) Toroidal pore model: accumulation of AMPs vertically embed in the cell membrane, and then, bend to
form a ring hole.

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Huan et al.
is high. Human cathelicidin LL-37 exhibits its activity through
this mechanism, and AMPs with β-sheet structure also play a
role in this model (Shenkarev et al., 2011; Corrêa et al., 2019).
Polarized light-attenuated total reflection Fourier transform
infrared spectroscopy (ATR-FTIR) was used to study the effect
of AMP cecropin P1 on the bacterial cell membrane and found
that it was an applied flat on the surface of the pathogen’s
cell membrane to destabilize and eventually destroy the cell
membrane (Lyu et al., 2019).
Membrane targeting mechanisms (the cell membrane
composition differences of bacteria and fungi shown in Figure 7)
can be further refined to address the large differences in the
lipid composition of the cell membranes of bacteria, fungi,
and mammals. The main lipids in cell membranes include
glycerophospholipids (GPLs), lysolipids, sphingolipids, and
sterols. Phosphatidylethanolamine (PE), phosphatidylglycerol
(PG), and cardiolipin (CL) are the most common anionic lipids in
bacteria, whereas phosphatidylcholine (PC), phosphatidylinositol
(PI), PE, and phosphatidic acid (PA) are the main GPLs in fungal
cell membranes (Ejsing et al., 2009; Singh and Prasad, 2011;
Li et al., 2017). Furthermore, fungal cell membranes are more
anionic than mammalian cell membranes and have higher PC
content. Meanwhile, ergosterol is the sterol found in the plasma
membrane of lower eukaryotes, such as fungi, whereas that of
animals contains cholesterol (Faruck et al., 2016). Many AMPs
take advantage of differences in membrane components to
exert their effects.
Antimicrobial peptides are promising to be anti-biofilm agents
but it should be noticed that they are different from the cell
penetrating peptides (CPPs) which typically comprise 5–30
amino acids and can translocate across the cell membrane. CPPs
could be categorized according to physicochemical properties
into three classes: Cationic, amphipathic, and hydrophobic,
but anti-biofilm peptides have stricter requirements for these
physicochemical properties. Anti-biofilm peptides target the
biofilms by different mechanisms including (1) degradation of
signals within biofilms; (2) permeabilize within cytoplasmic
FIGURE 7 | Comparison of Gram-negative bacteria, Gram-positive bacteria and fungi cell walls.
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Antimicrobial Peptides: Classification, Design, Application
membrane/EPS; (3) modulating EPS production etc. and then
can address chronic multi-resistant bacterial infections (Pletzer
et al., 2016; Ribeiro et al., 2016; Guidotti et al., 2017;
Derakhshankhah and Jafari, 2018; Rajput and Kumar, 2018). For
instance, SAAP-148, synthesized based on LL-37, showed activity
to prevent biofilm formation by S. aureus and A. baumannii
(Crunkhorn, 2018).
Non-membrane Targeting Mechanism
The way of AMPs entering cells is direct penetration or
endocytosis. After entering the cytoplasm, AMPs will identify and
act on the target. Depending on the target, AMPs can be divided
into the following categories.
Inhibition of Protein Biosynthesis
Antimicrobial peptides affect transcription, translation, and
assembly into functional peptides through molecular chaperone
folding by interfering with related enzymes and effector
molecules. For example, Bac7 1–35 targets ribosomes to inhibit
protein translation (Mardirossian et al., 2014), whereas Tur1A
inhibits protein synthesis in E. coli and Thermus thermophilus by
inhibiting the transition from the initial phase to the extension
phase. However, the differences between Tur1A and Bac7 also
lead to various ways of binding to ribosomes and interacting
with the ribosomal peptide exit tunnel (Mardirossian et al.,
2018). But some AMPs’ have different targets. For instance,
genome-wide transcription shows that the AMP DM3 can affect
many important intracellular pathways of protein biosynthesis
(Le et al., 2016).
Chaperones are key proteins for correctly folding and
assembling newly synthesized proteins and make them have
stereoisomerism, which makes AMPs have cell selectivity and
can prevent cytotoxicity. According to a previous review: both
pyrhocoricin and drosocin can prevent DnaK from refolding
misfolded proteins by inducing a permanent closure of the
DnaK peptide-binding cavity (Kragol et al., 2001; Le et al., 2017;
Wrońska and Boguś, 2020).
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Inhibition of Nucleic Acid Biosynthesis
Antimicrobial peptides can affect key enzymes or induce the
degradation of nucleic acid molecules to inhibit nucleic acid
biosynthesis. Indolicidin, a C-terminal-amidated cationic Trp-
rich AMP with 13 amino acids, specifically targets the abasic site
of DNA to crosslink single- or double-stranded DNA and it can
also inhibit DNA topoisomerase I (Subbalakshmi and Sitaram,
1998). TFP (Tissue factor pathway inhibitor)1-1TC24, which is
an AMP from tongues, enters the cytoplasm of target cells after
the rupture of cell membrane and then degrades DNA and RNA
(He et al., 2017).
Inhibition of Protease Activity
Many AMPs can inhibit various metabolic activities by inhibiting
protease activity. For example, histatin 5 has a strong inhibitory
effect on the proteases secreted by the host and bacteria.
AMPs eNAP-2 and indolicidin inhibit microbial serine proteases,
elastase, and chymotrypsin (Le et al., 2017). Cathelicidin-BF
is a peptide isolated from the venom of Bungarus fasciatus, it
can effectively inhibit thrombin-induced platelet aggregation and
further block protease-activated receptor 4 (Shu et al., 2019).
Inhibition of Cell Division
Antimicrobial peptides inhibit cell division by inhibiting
DNA replication and DNA damage response (SOS
response), blocking the cell cycle or causing the failure of
chromosome separation (Lutkenhaus, 1990). For instance, APP
(GLARALTRLLRQLTRQLTRA), which is an AMP with 20
amino acid residues, can efficiently kill C. albicans because of
its cell-penetrating efficiency, strong DNA-binding affinity, and
ability to induce S-phase arrest in intracellular environment
(Li L. et al., 2016). MciZ, which has 40 amino acid residues, is
an effective inhibitor of bacterial cell division, Z-ring formation,
and localization (Cruz et al., 2020).
Moreover, it has been reported that several AFPs
have damaging effects on the organelles of fungi. For
example, Histintin 5 can interact with mitochondria,
causing the production of ROS, and inducing cell death
(Helmerhorst et al., 2001).
In addition to intracellular targets, differences in cell wall
composition, such as lipopolysaccharide (LPS), lipid A and
mannoproteins, are potential targets for AMPs. Specifically,
Gram-positive and Gram-negative bacteria are classified based
on their bacterial cell wall structure. Gram-positive bacteria have
a layer of cross-linked peptidoglycan, whereas Gram-negative
bacteria have an additional outer membrane with an inner
leaflet containing only phosphatidic acid and an outer leaflet
made of LPS. LPS has numerous negatively charged phosphate
groups, which combine with a salt bridge with a divalent
cation (e.g., Ca2+ and Mg2+ ) to form an electrostatic network
(Nikaido, 2003). This electrostatic zone is the main barrier
against hydrophobic antibiotics and causes the low permeability
of Gram-negative bacteria. The main components of the fungal
cell wall are mannoprotein, β-glucans and chitin (polymers of
1,4-β-N-acetylglucosamine) and the mutations in the relevant
genes of the LPS pathway and phospholipid trafficking provide
resistance to the AMPs (Cabib, 2009; Spohn et al., 2019).
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Antimicrobial Peptides: Classification, Design, Application
Mannoproteins in fungal cell walls include a variety of proteins,
including structural proteins, cell adhesion proteins (floccrin
and lectin) and enzymes involved in cell wall synthesis and
remodeling (hydrolytic enzymes and transglycosylase). These
proteins differ from human cell membrane proteins and are
potential targets of AFPs (Rautenbach et al., 2016). Furthermore,
teichoic acid and lipoteichoic acid in the cell wall are also
potential targets of AMPs and these theories could support the
design of AMPs with low cytotoxicity.
DESIGN METHODS OF ANTIMICROBIAL
PEPTIDES
Antimicrobial peptides have good application prospects.
However, AMPs have the following problems. (1) AMPs damage
the cell membrane of eukaryotes and cause hemolytic side effects;
(2) rising production costs and technical problems limit their
manufacture; (3) their stability is limited at certain pH; (4) AMPs
have reduced activity under the presence of iron and certain
serum; (5) AMPs are easily hydrolyzed by proteases. Therefore,
the ideal AMP should meet the following characteristics:
(i) high antimicrobial activity; (ii) low toxicity to mammalian
membranes; (iii) high protease and environment stability;
(iv) low serum binding capacity and (v) ease of access and low
cost production (Li et al., 2017). Therefore, designing AMPs
to achieve the desired effect has attracted increasing attention.
The rational design of antibacterial peptides should focus on
the following five aspects: chain length, secondary structure, net
charge, hydrophobicity, and amphiphilicity and these have been
mentioned in many studies and this review will focus more on
several specific methods of antimicrobial peptide design.
Site-Directed Mutation
Site-directed mutation refers to the redesign of natural
antimicrobial peptides by adding, deleting or replacing one, or
several amino acid residues (Torres et al., 2019).
De novo Design Peptides
The de novo design of peptides attaches importance to the
design of amphiphilic AMPs (Guha et al., 2019). For example,
GALA is a well-known de novo-designed AMP. Amphipathic
α-helical peptide GALA is created by placing protonatable
glutamic acid residues in most positions with the spacing of i
to i + 4 (Goormaghtigh et al., 1991). The repeated sequence
(XXYY)n, where X 1 and X 2 are hydrophobic amino acids, Y 1
and Y 2 are cationic amino acids, and n is the number of
repeat units, is designed based on the hydrophobicity cycle that
mimics natural α-helical AMPs and successfully designs broad-
spectrum α-helical AMPs. Sequences (LKKL)3 and (WKKW)2.5
have the highest selectivity (Khara et al., 2017). Moreover,
Ll Km W2 model peptides are also de novo-designed peptides.
Amphipathic helical properties were conferred by using leucines
and lysines, and two tryptophan residues were positioned at
the amphipathic interface between the hydrophilic ending side
and the hydrophobic starting side. Among the model peptides,
L4 K5 W2 has good anti-MRSA activity (Lee et al., 2011).
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Template-Based Design Method
Sequence templates can be obtained by comparing a large
number of structurally homologous fragments of natural
AMPs (such as HDPs) and extracting conservative patterns
based on the type of residue (such as charged, polar,
hydrophobic, etc.) (Zelezetsky and Tossi, 2006). Based on
the modification, the parameters, such as helix formation
tendency, cationic, amphiphilicity and overall hydrophobicity,
can be systematically changed. For instance, cecropin, magainin,
protegrin, and lactoferrin have all been used as AMP templates
(Fjell et al., 2012).
Based on the Self-Assembly of
Antimicrobial Peptides
Peptides can form nanostructures, such as micelles, vesicles,
nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and
can increase or impart antibacterial activity to AMPs during
the self-assembly of peptides. For example, KLD-12 (KLD) is
a self-assembling peptide with 12 amino acid residues that can
adopt nanostructures and are known for their tissue engineering
properties. The addition of Arg residues in KLD shows no
remarkable change in its β-sheet secondary structure and
the self-assembly characteristics of the forming nanostructures
(Tripathi et al., 2015). Dimer structure can also be used to
enhance the antimicrobial activity of AMPs and reduce toxicity,
but membrane-destabilizing effects are reduced after dimer
formation (Malekkhaiat Häffner and Malmsten, 2018).
Chemical Modification
Various chemical modifications of AMPs, including residue
phosphorylation, the addition of D-amino acids or unnatural
amino acids (homoarginine), cyclization, halogenation,
acetylation, and peptidomimetics, have been used to improve
the stability of peptides against proteases. Given that the enzyme
is stereospecific, the incorporation of unnatural D-amino acids
into the AMP sequence can reverse the stereochemistry and
prevent protease degradation (Zhong et al., 2020). The so-called
peptidomimetics, whose main elements mimic the structure of
peptides, are usually produced by modifications, such as chain
extension or heteroatom incorporation of existing peptides
(Patch and Barron, 2002). Ornine, which is an unnatural residue
with a positive charge and has a high resistance to protease
activity, is also used in non-chemical modification. Replacing Trp
residues with family residues, such as β-dihydrophenylalanine,
can stabilize secondary structures and improve antibacterial
properties (Maurya et al., 2013).
Halogenation
Halogenation is highly related to the activity, specificity, and
stability of AMPs. In the latest report, Halogen is introduced into
jelleine-I which is a short peptide isolated from the royal jelly
of honeybees (Apis mellifera) by replacing phenylalanine with
a halogenated phenylalanine analog, increasing the antibacterial
activity in vitro and anti-biofilm activity. In addition, the
proteolytic stability of jelleine-1 is increased by 10–100
times by halogenation (Jia et al., 2019). The halogenated
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Antimicrobial Peptides: Classification, Design, Application
peptidomimetic α,α-disubstituted β-amino amides are also
promising bacteriostatic drugs that have inhibitory effects on
more than 30 multi-resistant clinical isolates of Gram-positive
and Gram-negative bacteria (Paulsen et al., 2019). Halogenation
is also related to the specificity of AMPs. The o-fluorine
substitution in phenylalanine residues maintains the activity of
temporin L on E. coli but leads to the loss of activity on S. aureus
and P. aeruginosa (Setty et al., 2017).
Cyclisation
Three modes of cyclisation, including cyclisation via disulfide
bonds, head-to-tail cyclisation and internal bonding between
side chains, have been found in natural AMPs. The synthesis of
disulfide bonds often complicates the development of synthetic
peptides. The circularisation of the main chain of arenicin-1
molecule resulted in increased activity against drug-resistant
clinical isolates but caused no substantial effect on cytotoxicity
(Orlov et al., 2019). The HDPs tachyplesins I, II, and III
and their cyclic analogs cTI, cTII, and cTIII, respectively,
have similar structures and activities and can resist bacterial
and cancer cells. The cyclisation of the backbone reduces the
hemolytic activity and improves the stability of the peptides
whilst maintaining effective anticancer and antibacterial activities
(Vernen et al., 2019).
Capping
Capping refers to the addition of specific motifs or modifications,
such as amidation at the C-terminus and acetylation at the
N-terminus, rendering AMPs with more natural peptide
characteristics. Post-translational modifications play an
important role in the function of AMPs and are the most
commonly used in peptide design. The C-terminal Rana
box (consisting of a C-terminal cyclic heptapeptide with a
conservative disulfide bond) and amide group are important
C-terminal capping methods. For example, the C-terminal
amide group of maximin H5 can enhance antibacterial efficacy
without increasing lytic ability (Dennison et al., 2015). The
N-terminal lipidated analog C4 VG16 KRKP shows enhanced
antibacterial activity against various Gram-negative bacteria.
The functions of N-terminal lipidation include (i) increasing
LPS neutralization, (ii) increasing stability to proteases and
peptidases, and (iii) reducing cytotoxicity (Datta et al., 2016).
Furthermore, hydrophobic end labeling is a commonly used
method to increase the activity of antimicrobial peptides. Acyl
lipid peptides have a linear or cyclic structure in which one or
more hydrocarbon tails are connected to the N-terminus of a
short oligopeptide (Chu-Kung et al., 2010). Lipopeptides have
covalently attached hydrophobic moieties, such as sterols or
fatty acids. Aromatic amino acid terminal labeling is also the
main hydrophobic terminal labeling method. Tryptophan (W)
and phenylalanine (F) are the commonly used aromatic amino
acids. Their large and polarisable residues have an affinity for
the interface, and the W/F tag is also sensitive to the differences
between ergosterol and cholesterol and can prevent self-assembly.
This condition results in low aggregation numbers and high
critical aggregation concentrations (Schmidtchen et al., 2014).
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Conjugation
Peptide conjugation has been the goal of most research in recent
years to produce active and stable AMPs with high selectivity.
Different side chains or AMP fragments can be used aside from
the repetition of the same amino acid motifs. For example,
conjugating fatty acids with a length of 8–12 carbon atoms to
the 4th or 7th side chain of the D-amino acids of Ano-D4,7
improves antibacterial selectivity and anti-biofilm activity. In
addition, the new peptide exhibits high stability against trypsin,
serum, salt, and different pH environments (Zhong et al., 2020).
The conjugation of different AMPs can also be performed. For
example, the hybrid peptide (PA2–GNU7) constructed by the
addition of PA2 to GNU7 has a high activity and specificity to
P. aeruginosa (Kim et al., 2020).
Synthetic Mimics of AMPs (SMAMPs)
SMAMPs include a broad family of molecular entities based on
the structure and function of AMPs. However, their backbones
are not entirely based on α-amino acids, including β-amino
acid oligomers, arylamide oligomers, and phenylene ethynylenes
(Michael Henderson and Lee, 2013). For instance, SMAMP10,
which is a potential drug for intravenous treatment, causes no
drug resistance and has a strong inhibitory effect on MRSA and
vancomycin-resistant Enterococcus faecium (Tew et al., 2006).
Peptoids
Peptoids are peptide isomers, in which the side chain is bonded
to the main chain nitrogen instead of α-carbon or poly-N-
substituted glycine in which the side chain is connected to
amide nitrogen instead of the α-carbon on the main chain
(Andreev et al., 2018). For example, the cationic peptide
SA4 (IOWAGOLFOLFO-NH2) and its poly-N-substituted
glycine homolog SPO (nInOnWnAnGnOnLnFnOnLnFnO-
NH2 ) inhibit the planktonic and biofilm formation of
A. baumannii strains, which are susceptible to multi-drug
resistance (Sharma et al., 2019).
Use of Motifs
Motifs with specific functions have been reported increasingly.
These motifs can be repeated units for combining into new
antimicrobial peptides, or specific amino acid combination
units appearing at the end (such as capping) of or even in
the peptide chain.
Motif at the End of the Chain
ATCUN motif
This motif includes two tripeptide structures, including Gly–Gly–
His or Val–Ile–His, which are added at the end of the peptide
chain. ATCUN-containing AMPs in the presence of hydrogen
peroxide and ascorbic acid combine with Cu2+ to induce the
valence of copper ions between +2 and +3 oxidation states
and form an ATCUN–Cu (II) complex, generating ROS by
Fenton-like reactions. Extracellular polymeric substances (EPS)
are important for biofilms and can enhance the resistance of cells
to antibacterial agents (Flemming, 2016). ATCUN–AMPs have
been used to degrade environmental DNA, which is one of the
major components of EPS. Several related practical applications
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Antimicrobial Peptides: Classification, Design, Application
have been reported. For example, the biological activity against
carbapenem-resistant Enterobacteriaceae is increased by adding
this motif to the N-terminus of an alpha-helical AMP (such as
CM15). Besides, the Cu–ATCUN derivative of OV-3 containing
a C-terminal GGC sequence showed high levels of membrane
permeation and lipid peroxidation. The concept of catalytic metal
drugs has attracted widespread attention although the concept is
still in its infancy because of the role of metal ions (Alexander
et al., 2017; Agbale et al., 2019).
Rana box
Rana box: Rana box is a heptapeptide motif (CGLXGLC) from
the nigrocin family. Rana box consists of two cysteine residues
that are separated by four or five other residues on the side and
can form a cyclic disulfide bond. Rana box peptide has shown
structural analogies with polymyxin (colistin), and the primary
structure of the Rana box motif is important in determining
bacteriostatic activity (Kozić et al., 2015). The deletion of the
‘Rana box’ motif will cause the AMP antibacterial effect to
disappear, but replacing the natural ‘Rana box’ sequence of
AMPs with amidated phenylalanine can expand its efficacy
against antibiotic-resistant microorganisms, including MRSA
and P. aeruginosa, and reduce cytotoxicity. This phenomenon
also shows that the effect of the motif on AMPs needs to be
determined based on the specific situation and is not completely
beneficial (Bao et al., 2018).
LPS binding motif
The LPS binding motif (G-WKRKRF-G) can produce a broad
spectrum of antibacterial activity when introduced into the
C-terminus of temporin-1 Ta and temporin-1 Tb (close isoforms
of temporin) (Mohanram and Bhattacharjya, 2016).
γ-core motif
Antifungal Peptides have a conserved GXC(X3−9 ) C γ-core motif
(residues 5–14, GKCYKKDNIC; d-isomer) at its N-terminus,
which is a cation part of the ring. This conserved motif interferes
with the integrity of the plasma membrane of the cell (Yount and
Yeaman, 2004). Conserved γ-core motifs are directly involved
in protein–membrane interactions and strongly contribute to
membrane binding (Utesch et al., 2018).
Motif in the Chain
If replace d-Phe1-Pro2 sequence in peptide chain with d-Phe-2-
Abz turn motif (2-Abz is an abbreviation of 2-aminobenzoic acid
D -amino acid) in AMP Tyrc A, and nuclear magnetic resonance
shows that this change retains the β-hairpin structure. Unlike
the traditional β-turn motif, the D-Phe-2-Abz motif can be used
as a tool for β-hairpin libraries. The hydrophobic peptide can
be formed into the nucleated β-hairpin formation by adding
the D-Phe-2-Abz motif. Moreover, the inclusion of this part in
two designed cationic amphiphilic peptides can produce broad-
spectrum antibacterial activity and low hemolysis rate (Cameron
et al., 2017; Cameron et al., 2018).
NGR motif
The NGR motif is composed of Asn–Gly–Arg, and AMPs with
this structure have strong cytotoxicity (Table 1). The data indicate
that the new AMPs containing NGR may bind to CD13+ or
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αvβ3+ tumor cells by binding to CD13 or αvβ3, respectively,
to exert anti-tumor activity, especially on CD13+ tumor cells
(Zhang et al., 2015).
“Glycine zipper” of GxxxG motifs
The central GxxxG motif can induce strong self-assembly and
have been already used in the design of AMPs (Brosig and
Langosch, 1998; Krauson et al., 2012).
Motif-Based Polyvalent Peptide Synthesis (Dimers,
Tetramers, etc.)
Bovine lactoferrin B is an AMP composed of 25 amino acid
residues and has antibacterial, antifungal, and antiparasitic
activities. The multivalent molecules LfcinB (20–25)2 and LfcinB
(20–25)4 contain the LfcinB (20–25) motif (RRWQWR) and
show inhibition activity against E. coli, P. aeruginosa, and
S. aureus. Chimeric peptide chimera 3 containing two motifs,
namely, the RRWQWR of LfcinB (20–25) and the RLLR of BFII
(32–35), shows high antibacterial activity against E. coli ATCC
25922 and S. aureus ATCC 25923 (Vargas-Casanova et al., 2019;
Pineda-Castañeda et al., 2020).
Computer Design
Computer design includes simple statistical modeling, Structure-
activity relationships study (Abdel Monaim et al., 2018), neural
networks (Müller et al., 2018), deep learning (Veltri et al., 2018),
word embedding (Hamid and Friedberg, 2018) and machine
learning. For example, a machine learning method by Matlab
is proposed based on the concept of scoring the contribution
of each amino acid’s antibacterial activity (Wu X. et al., 2014).
The genetic algorithm was used to design the amphiphilic
α-helical peptide guavalin 2, which has an uncommon amino
acid composition (three tyrosine and three glutamine residues)
and interestingly causes membrane hyperpolarization, which
is a different mechanism from those of other AMPs (Porto
et al., 2018). Two research methods have been developed based
on the research background of quantitative structure–activity
relationships: prediction method based on AMP therapeutic
index and the identification of novel potential AMPs from the
expressed sequence tag database based on the principles of
the highly conserved signal peptide subclasses related to AMPs
(Juretić et al., 2011).
TABLE 1 | Examples of AMPs with NGR motif.
Name Sequence 3D structure
CORTICOSTATIN I ICACRRRFCPNSERFSGY Bridge
CRVNGARYVRCCSRR
NP-3a GICACRRRFCPNSERFSGY Bridge
CRVNGARYVRCCSRR
Corticostatin VI GICACRRRFCLNFEQFSG Bridge
YCRVNGARYVRCCSRR
Pediocin PA-1/AcH KYYGNGVTCGKHSCSVDWGKA Combine Helix and
TTCIINNGAMAWATGGHQGNHKC Beta structure
Lacticin 3147 CSTNTFSLSDYWGNNGA Helix
WCTLTHECMAWCK
As-CATH5 TRRKFWKKVLNGALKIAPFLLG Helix
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Antimicrobial Peptides: Classification, Design, Application
Rational Library Design
In this way, a variety of AMP variants can be obtained. If
combined with high-throughput screening, it can effectively
obtain the desired AMP. For instance, some new AMPs are
designed by the combinatorial peptide library of melittin and
show higher activity and lower cytotoxicity (Krauson et al., 2015).
ENVIRONMENTAL FACTORS
AFFECTING THE ACTIVITY OF
ANTIMICROBIAL PEPTIDES
Metal Ions
Cations, such as Na+ and Mg2+ , may affect AMP activity
(Zhu et al., 2015). However, the different valences of metal
ions have varied effects on AMPs. For example, divalent cations
show stronger antagonism to bacteria than monovalent cations
with thanatin and s-thanatin, which are insect AMPs (Wu
et al., 2008). In the presence of NaCl, the signal response
during the association phase remarkably decreased in single-cycle
and multi-cycle kinetic experiments, resulting in a decreased
association rate. This occurrence may be caused by the shielding
effect of NaCl between the cationic peptide and the zwitterionic
membrane. Another possible reason is that Na+ can bind to the
phospholipid bilayer, where the ions interact with the phosphate
and the carbonyl oxygen of lipid head groups (Sabapathy et al.,
2020). The reduced activity of synthetic peptide [RLLR]5 under
high salt concentration is possibly caused by the destruction of its
α-helix structure.
Table 2 shows that several AMPs, including histatin,
myxinidin, and hepcidin, contain ATCUN motifs (Amino
Terminal Copper and Nickel with XXH sequence). Iron is the
most abundant metal ion in human saliva, but the combination
with this metal ion results in the loss of the α-helix of histatin
5 and greatly reduces its antifungal activity (Puri et al., 2015).
However, the coordination of copper (II) and nickel (II) ions can
induce the formation of ROS, which is essential for bactericidal
activity (Jeżowska-Bojczuk and Stokowa-Sołtys, 2018).
Anionic AMPs have a large number of negatively charged
aspartic and glutamic acid residues (Lakshmaiah Narayana and
Chen, 2015). They require zinc as a functional cofactor and the
Activity Reference
Anti-Gram+ and Gram− Zhu et al., 1988
Anti-Gram+ and Gram−, Antiviral, and Antifungal Selsted et al., 1985
Anti-Gram+ and Gram− Fuse et al., 1993
Anti-Gram+, Spermicidal Henderson et al.,
1992
Anti-Gram+, Spermicidal Martin et al., 2004
Anti-Gram+ and Gram−, Antifungal, anti-sepsis Chen Y. et al., 2017
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TABLE 2 | Effect of metal ions on AMPs with ATCUN motif activity.
Name Source Sequence Details
Histatin 5 Human parotid DSHAKRHHGYKR It losses the α-helical structure
saliva KFHEKHHSHRGY by binding iron and
coordination of copper (II) and
nickel (II) ions induces the ROS.
Myxinidin Epidermal mucus GIHDILKYGKPS It is similar to that of other
of hagfish peptides with the ATCUN motif.
Hepcidin Human liver DTHFPICIFCCGC When in the presence of
25 CHRSKCGMCCKT copper (II) ions and an
intracellular substance such as
ascorbate, hepcidin 25 may
generate ROS.
zinc complex shows stronger antibacterial activity (Jiang et al.,
2014). Several of these AMPs use metal ions to form cationic salt
bridges with the negatively charged components of the microbial
membrane to penetrate the membrane. Anionic AMPs may
attach to ribosomes or inhibit ribonuclease activity when in the
cytoplasm (Jeżowska-Bojczuk and Stokowa-Sołtys, 2018).
Metal ions also affect the self-assembly of peptides. These ions
can recognize specific amino acids, such as lysine and glutamic
acid, and may form salt bridges between peptide molecules to
induce peptide self-assembly. For example, Zn2+ can stabilize the
aggregation of peptides on the cell membrane, which results in
the enhanced antibacterial effect of DCD-1L in the presence of
Zn2+ (Tian et al., 2015).
pH
Many AMPs are stable and retain their antimicrobial activity
in a wide pH range. AMPs have enhanced activity at low
pH because of their basic properties. This condition is related
to the protonation of histidine at acidic pH, which promotes
electrostatic interactions with anionic surfaces, including LPS
and the anions of phospholipids, and subsequently enhances
antibacterial properties. The effect of pH on the antibacterial
activity of AMPs varies. For example, thanatin’s activity at
neutral pH is slightly higher than that under acidic conditions.
By contrast, the activity of xylan on E. coli, Listeria, and
C. albicans is remarkably higher at pH 5.5 than at pH 7.4
(Holdbrook et al., 2018). The inactivation of the histidine-
containing AMP C18G-His under low pH conditions involves
pH-dependent changes in the state of the aggregates in the
solution, because the aggregates, which are sensitive to pH and
lipid composition, may be affected by binding and conformation.
Peptides can also enhance bacterial membrane permeability at
low pH (Hitchner et al., 2019). Thrombin-derived C-terminal
peptides (TCPs) will also change the mode of CD14 (a protein
that is abundant in human plasma) from anti-inflammatory
mode to bacterial elimination mode from pH 7.4 to pH 5.5
(Holdbrook et al., 2018). A dimer (e.g., P-113) can be created
to provide AMPs with resistance to a higher pH range. The
sensitivity of this pH-sensitive AMP can be used to achieve
a certain targeting effect in practical applications. In addition,
charge interaction is one of the most important factors in peptide
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Antimicrobial Peptides: Classification, Design, Application
self-assembly. pH affects the charge state of amino acid and
substituent functional groups. Therefore, adjusting the pH is
the most common method for controlling peptide assembly and
disassembly (Tian et al., 2015).
Proteases
Proteases have a strong destructive effect on AMPs. For instance,
LL-37, which has the strongest inhibitory effect on chlamydial
infection, is inhibited by the protease chlamydial protease-like
activity factor (CPAF) secreted by Chlamydia (Tang et al., 2015).
Studies have been focused on the design of AMP carriers to solve
this problem (Lewies et al., 2017; Nordström et al., 2019). The
presence of chitosan–silica solid support of KR-12 peptide can
protect it be hydrolyzed by α-trypsin, and the degree of protection
is increased by 38% compared with the free KR-12 (Diosa et al.,
2020). However, several enzymes, such as protease 65, esterase 66
and phosphatase 67, cut the blocking group of the peptide and
trigger the self-assembly of the peptide, which positively affects
AMPs (Tian et al., 2015).
CURRENT PROGRESS AND
APPLICATION OF ANTIMICROBIAL
PEPTIDES
Medicine
Antimicrobial peptides can regulate pro-inflammatory reactions,
recruit cells, stimulate the proliferation of cells, promote
wound healing, modify gene expression and kill cancer cells
to participate in the immune regulation of human skin,
respiratory infections, and inflammatory diseases (de la Fuente-
Núñez et al., 2017). For example, α-defensins HNP-1, HNP-2,
and HNP-3 showed effective antibacterial activity against
adenovirus, human papilloma virus, herpes virus, influenza virus
and cytomegalovirus. Pulmonary diseases, such as idiopathic
pulmonary fibrosis, alveolar proteinosis, and acute respiratory
distress syndrome, show elevated levels of AMPs (Guaní-Guerra
et al., 2010). Likewise, AMPs secreted by the Paneth cells in
the mammalian gut are important to shape the gut microbiota
(Bevins and Salzman, 2011).
The application of AMPs in medicine, such as dental, surgical
infection, wound healing and ophthalmology is developing now.
But there are only three AMPs that have been approved by FDA
including gramicidin, daptomycin, and colistin.
Dental caries, endodontic infections, candidiasis, and
periodontal disease are common diseases in the human oral
cavity. Dental caries is a prevalent oral disease and some
acidogenic bacteria like Streptococcus sp. are the main caries-
associated pathogens (Izadi et al., 2020). Several AMPs have
good application potential. For instance, peptide ZXR-2
(FKIGGFIKKLWRSLLA) has shown potent activities against
pathogenic bacteria of dental caries, Streptococcus mutans,
Streptococcus sobrinus, and Porphyromonas gingivalis and
peptide PAC-113 (Clinical trial identifier: NCT00659971) that
has been sold over the counter in Taiwan for treating oral
candidiasis (Chen L. et al., 2017).
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Huan et al.
In surgical infection and wound healing: surgical infection
occurs after surgery, burns, accidental injury, skin disease, and
chronic wound infections have a serious hazard to human life
(Thapa et al., 2020). Several AMPs have shown the therapeutic
potential of these diseases. For example, AMP PXL150 shows
pronounced efficacy as an anti-infective agent in burn wounds
in mice and AMP D2A21 has been in the third phase of clinical
trials for treating burn wound infections (Björn et al., 2015).
In ophthalmology: Human eyes are prone to be infected
by several organisms including bacteria and fungi in which
S. aureus, Streptococcus pneumoniae, P. aeruginosa, Aspergillus
spp., and C. albicans are the most relevant pathogens (Silva
et al., 2013). Although AMPs such as Lactoferricin B, Protegrin-1
exhibited antimicrobial activity against these pathogenic bacteria,
their application in the field of ophthalmology is only at the
theoretical stage. With the popularity of contact lenses and the
increase in cases of related eye infections, antimicrobial peptides
have shown good application prospects in ophthalmology
(Khan and Lee, 2020).
Additional methods need to be performed for the application
of AMPs as drugs in medicine. The main strategies include
(1) constructing precursors to reduce cytotoxicity and improve
protease stability, (2) using AMPs in combination with
existing antibacterial agents, (3) inducing the correct expression
of AMPs with appropriate drugs and using engineering
probiotics as vectors to express AMPs. For example, in the
field of wound repair, different formulation strategies, such
as loading AMPs in nanoparticles, hydrogels, creams, gels,
ointments, or glutinous rice paper capsules, have been developed
to effectively deliver AMPs to the wound (Borro et al.,
2020; Thapa et al., 2020). In recent research, the sponges
developed from modified starch and HS-PEG-SH are covalently
immobilized with AMP showed effective antibacterial activity
(Yang et al., 2019).
More technical means, including pheromone-labeled AMPs,
local environment-triggered AMPs (enzyme precursor drug
release system, pH-activated AMPs, etc.), have been developed
to improve the targeting mechanism of AMPs. Furthermore,
nanotubes, quantum dots, graphene, and metal nanoparticles
have been proposed to be a potential method to enhance
drug delivery of AMPs (Magana et al., 2020). Hybrid peptides
have also been used to build targeting peptides. For example,
PA2, which is a P. aeruginosa-targeting peptide, was combined
with GNU7 (a broad-spectrum AMP) to construct a hybrid
peptide (PA2–GNU7) that targets OprF protein and has
good bactericidal activity and specificity (Kim et al., 2020).
Furthermore, some antibiotics, for instance, daptomycin (a
lipopeptide), lugdunin which is a 21-membered cyclic peptide
consists of 6 amino acid residues plus a thiazolidine moiety
and telavancin (a glycopeptide) have been widely used for
the clinic (Durand et al., 2019; Lampejo, 2020). Although
they are antibiotics, they have provided broader ideas for
the design of AMPs.
Food
Food preservatives have potential harm to the human body.
Therefore, natural preservatives are being advocated by more
Frontiers in Microbiology | www.frontiersin.org
Antimicrobial Peptides: Classification, Design, Application
people. AMPs have a good inhibitory effect on common bacteria
and fungi in food, and many AMPs are resistant to acids,
alkalis, and high temperatures are easily hydrolyzed by proteases
in the human body. Thus, AMPs are a promising alternative
to preservatives. Nisin is a bacteriocin produced by L. lactis
subspecies. Lactic acid bacteria have been widely used as food
preservatives. Nisin is categorized as generally recognized as safe
(GRAS) by the US Food and Drug Administration (FDA) and
is used as a food preservative in other countries (Khan and Oh,
2016). However, only nisin and polylysine are currently approved
by the FDA as food additives (Santos et al., 2018). Pedocin
PA-1, a bacteriocin consisting of 44 amino acids produced by
a diplococcus, is also used as a food preservative and is sold
on the market under the trade name ALTA 2431. Pedocin
PA-1 is used as a food additive to inhibit the growth of
L. monocytogenes, which can cause meat deterioration (Settanni
and Corsetti, 2008). Enterocin AS-48 is an AMP used to preserve
cider, fruit and vegetable juices, and enterocin CCM4231 is
used to preserve soy milk (Rai et al., 2016; Santos et al., 2018).
Encapsulating bacteriocins into liposomes is a new method used
to overcome the problems of AMPs in food applications (such
as proteolytic degradation or interaction with food ingredients)
(da Silva Malheiros et al., 2010).
Moreover, active packaging by adding AMPs is a novel
packaging method that has great potential in the food industry.
For instance, ε-poly-L-lysine is used in conjunction with starch
biofilms to show good inhibitory effects on Aspergillus parasiticus
(aflatoxin producer) and Penicillium expansum and nisin have
the potential to be dairy preservative because it is a highly
surface-active molecule (Luz et al., 2018).
Animal Husbandry and Aquaculture
The European Union banned the use of animal growth promoters
in animal feed in 2006. Thus, a new antibacterial strategy is
needed. Many AMPs are the potential to be used in poultry,
swine, and ruminants breeding and aquaculture because they
can improve production performance (Liu et al., 2008; Bao
et al., 2009), immunity and promote intestinal health and
some of them have a stronger inhibitory effect on bacterial
inflammation if used with antibiotics (Wang et al., 2019; Cote
et al., 2020). For example, SIAMP has a good effect on the
treatment of IBV in chicken (Sun et al., 2010). By adding swine
gut intestinal antimicrobial peptides (SGAMP), broilers showed
higher average daily gain and feed efficiency under chronic heat
stress conditions (Hu et al., 2017). Frog caerin 1.1, European
sea bass dicentracin and NK-lysine peptides (NKLPs) have
good inhibitory effects on Nodavirus, Septicaemia haemorrhagic
virus, Infectious pancreatic necrosis virus and Spring viremia
carp virus, which are devastating to fish farming (León et al.,
2020). The AMP in soybean meal fermented by B. subtilis E20
effectively inhibits V. parahaemolyticus and Vibrio alginolyticus
and enhances the resistance level of Litopenaeus vannamei against
V. parahaemolyticus when added to feeds (Cheng et al., 2017).
Agriculture
For agriculture, the plant pathogenic infection of bacteria and
fungi causes the loss of economy, for instance, Aspergillus
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Huan et al. Antimicrobial Peptides: Classification, Design, Application

TABLE 3 | Application examples of AMPs in various fields.

AMPs/Product name Description Treatment/effect Company/reference

Medicine Dalbavancin (BI397, Semisynthetic lipoglycopeptide Acute bacterial skin infections Approved
Dalvance, Xydalba)
PAC-113, P-113 Histatin 5 derivative (12 amino acids) Oral candidiasis Phase IIb complete (sold
over the counter in Taiwan
by General Biologicals
Corporation)
Fuzeon Enfuvirtide HIV-1 infection Approved
Baciim Bacitracin Localized skin and eye infections, wound infections Approved
Vancocin Vancomycin Bacterial infections Approved
Daptomycin Lipopeptide Gram-positive infections Approved
Telavancin Glycopeptide (lipoglycopeptide) Skin infections, osocomial pneumonia Approved
Colistin Polymyxin E MDR infections caused by Gram-negative bacteria Approved
Gramicidin Cationic cyclic deca-peptide Purulent skin disease Approved
D2A21 Synthetic peptide Burn wound infections Phase III/Demegen
PXL01 Lactoferrin analog Postsurgical adhesions Phase III/ProMore Pharma
Omiganan (CLS001) Papulopustular rosacea; Phase III
Food Nisin Dairy (Listeria monocytogenes and Staphylococcus Approved
aureus)
Polylisine Natural cationic antibacterial agent Sushi, boiled rice, noodles, meat, and drinks Approved
Husbandry SGAMP Swine gut intestinal antimicrobial peptides Heat stress Hu et al., 2017
NKL-24 Zebrafish NK-lysin V. parahaemolyticus infection in the scallop Shan et al., 2020
Caerin1.1 Magnificent tree frog L. garvieae, porcine epidemic diarrhea virus (PEDV) León et al., 2020
Dicentracin European sea bass L. garvieae, viral hemorrhagic septicaemia (VHSV), León et al., 2020
infectious pancreatic virus (IPNV)
Agriculture PAF26 RKKWFW Green mold Wang et al., 2018
O3TR/C12O3TR H-OOWW-NH2/C12 -OOWW-NH2 P. digitatum Li X. et al., 2019
Thanatin Podisus maculiventris Rice blast disease, Sour rot (Geotrichum Imamura et al., 2010
(GSKKPVPIIYCNRRTGKCQRM) citri-aurantii)
Ponericin W1 Pachycondyla goeldii M. oryzae, Botrytis cinerea, and Fusarium Orivel et al., 2001
(WLGSALKIGAKLLPSVVGLFKKKKQ) graminearum, Sour rot (Geotrichum citri-aurantii)
Mastoparan-S Sphodromantis viridis G+, G−, Aspergillus niger, Aspergillus fumigates, Zare-Zardini et al., 2015
(LRLKSIVSYAKKVL) Sour rot (Geotrichum citri-aurantii)

flavus infection of corn and peanuts, citrus green mold
caused by Penicillium digitatum, gray mold disease caused
by Botrytis cinerea on strawberries and Geotrichum citri-
aurantii infection of citrus fruit all cause great harm to
the growth and post-harvest of agricultural products (Liu
et al., 2007; Liu et al., 2019). Several AFPs have shown
prospect to control these problems. However, the practical
application of antimicrobial peptides in the transportation
and preservation of agricultural products is still lacking,
because the use of antimicrobial peptides will greatly
increase the cost in the transportation of fruits and vegetables
(application examples of AMPs in these four fields are shown
in Table 3).
CONCLUSION
Antimicrobial peptides constitute a global research hotspot,
but many key issues in design and application need to
be solved urgently. Several restrictive factors hinder the
application of AMPs. The interaction of multidisciplinary
subjects, such as biology, materials science, chemistry,
bioinformatics, molecular informatics and pharmacy can
further develop prospective AMPs. Computer molecular
dynamics simulation, cell membrane simulation, and more
methods are being applied to study the mechanism of
AMPs. How to further understand the correlation between
AMPs and various targets instead of conducting one-sided
experimental research might improve experimental designs
to obtain stronger systemic and scientific demonstrations.
On this basis, further animal experiments are required
instead of simple cell-level experiments to test the
effect of AMPs under complex physiological conditions.
Several complicated methods, such as the chemical
method of peptidomimetics and non-natural amino acid
modifications, have been applied in designing AMPs to
solve the problem of protease hydrolysis. Most methods
use chemical substrates, but the cost of these methods
cannot be ignored in practice. In addition, chemical
synthesis and the use of engineered bacteria are currently
the mainstream for such procedures. Finding a better biological
preparation method, reducing the cost and increasing

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Huan et al.
the yield is important problems in practical application.
Furthermore, studying the AMP expression of the organism
itself and finding a better expression vector are necessary for
mass production in the future as more AMPs in nature are
discovered. Further research is needed on the reported AMPs
to solve the problem on structure–function relationship. As
a branch of peptide drugs, AMPs need to progress with the
advancement of medical science against the background of
the current low success rate of the clinical application of
AMPs. More attention can be focused on food, agriculture, and
animal husbandry.
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Conflict of Interest: The authors declare that the research was conducted in the
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